Cutinase cleaning compositions

ABSTRACT

This invention relates to cleaning compositions and methods for using them. Particularly, the invention relates to compositions comprising a surfactant and a cutinase enzyme. A preferred cutinase is derived from Pseudomonas putida ATCC 53552. Excellent cleaning is obtained with a surfactant mixture containing sodium dodecyl sulfate and octoxynol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is derived from PCT Application Ser. No.PCT/US88/01844, filed on May 31, 1988 which, in turn, is acontinuation-in-part of Ser. No. 07/056,500 and claims priority under 35U.S.C. 120 from U.S. Ser. No. 07/056,500, filed May 29, 1987 and whichis now abandoned.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to enzymatic cleaning compositions and methods forusing them. Particularly the invention relates to cleaning compositionscomprising a surfactant and a cutinase enzyme.

(b) Background Information

A wide variety of enzymes are well known for use in cleaningcompositions. The use of B. subtilisins and B. licheniformis protease incommercial preparations is common. Other enzymes have also been used incommercial cleaning compositions such as, for example, U.S. Pat. No.4,011,169, and British Patent No. 1,293,613. Also a comprehensive reviewarticle of lipases in cleaning compositions can be found in Journal ofApplied Biochemistry, 2:218-229 (1980) in an article entitled "Lipasesas Detergent Components". Lipolytic detergent additives are also knownfrom e.g., British Patent Specification No. 1,293,613 and CanadianPatent No. 835,343.

U.S. Pat. No. 3,950,277 and British Patent Specification No. 1,442,418disclose lipase enzymes combined with an activator and calcium and/ormagnesium ions, respectively, which are utilized to pre-soak soiledfabrics and to remove triglyceride stains and soils from polyester orpolyester/cotton fabric blends, respectively. Suitable microbial lipasesfor use therein (apart from animal and plant derived lipases) are saidto be those derived from Pseudomonas, Aspergillus, Pneumococcus,Staphylococcus, and Staphylococcus toxins, Mycobacterium tuberculosis,Mycotorula lipolytica, and Sclerotinia.

British Patent Specification No. 1,372,034 discloses a detergentcomposition comprising a bacterial lipase produced by Pseudomonasstutzeri strain ATCC 19154. Furthermore, it is recommended that thepreferred lipolytic enzymes should have a pH optimum between 6 and 10,and should be active in said range, preferably between 7 and 9. Around1970, this presumed Pseudomonas stutzeri strain was reclassified asPseudomonas aeruginosa, as appears for example from the ATCC catalogues.

European Patent Application EP-A-No. 0130064 discloses an enzymaticdetergent additive comprising a lipase isolated from Fusarium oxysporumwith an alleged higher lipolytic cleaning efficiency than conventionallipases.

In European Patent Application No. 0214761, enzymatic detergentadditives are described as the active component including a microbiallyproduced lipase from a strain of Pseudomonas cepacia. The lipasesdescribed therein are claimed to be superior to the lipolytic detergentaction of the prior art, especially at low temperature washing processes(around 60° C. and below).

In PCT Patent Application No. 87/00859 other novel lipolytic enzymes aredescribed as having an optimal pH in the range of 8-10.5 at atemperature of 60° C. or less from bacterial strains selected fromPseudomonas pseudoalcaligenes, P. stutzeri and Acinetobactercalcoaceticus. These enzymes are described as particularly effective atlow temperatures; i.e., 40° C. or lower and effective in both liquid andsolid detergent compositions.

Also in U.S. Pat. No. 3,950,277, it is described in general terms thatlipases from Pseudomonas are suited as agents for removal of oily stainsfrom fabrics, if used together with a special group of lipaseactivators. The art cited does not, however, cover cutinase enzymes fromPseudomonas or any other microbial source. However, prior art enzymesfor use in cleaning compositions, while effective on many proteins andlipids, are not completely effective against all stains commonly foundin laundry and other cleaning applications. Further, many lipases arenot stable at pH 8-11 where most cleaning compositions are used. Evenfurther, most enzymes for use in cleaning compositions are not verystable, if at all, under oxidative conditions or in the presence ofother enzymes such as proteases.

SUMMARY OF THE INVENTION

Accordingly it has been discovered that combinations of a surfactant anda substantially pure microbial cutinase enzyme are effectivecompositions for cleaning applications. The cutinase enzyme preparationspossess activity at pH of from about 8 and 11, exhibit cleaning activityin aqueous solution at concentrations from about 0.05 mg/L to about 100mg/L or m ore at temperatures from about 20° C. to about 50° C.

The enzymes are oxidatively stable and stable in the presence of otherenzymes such as proteases. Even further, the cutinases show asynergistic effect when a plurality of surfactants are used with thecutinase.

The invention also relates to the improved process for enzymaticallycleaning a material with an aqueous solution; the improvement comprisingadding a substantially pure cutinase to the cleaning solution.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have discovered that cutinase enzymes are useful whenincluded in cleaning compositions. These compositions may take on avariety of forms such as for laundry cleaning, household and industrialcleaning, and the like. The cleaning compositions comprise combinationsof known surfactants and a microbial cutinase enzyme which can be usedto clean a wide variety of materials. The composition can be added toaqueous solution or solid powder, or formulated in an aqueous solutionor solid powder and used according to conventional cleaning techniques.In one embodiment, the surfactant employed in combination with theselected cutinase is compatible with the cutinase.

Enzyme

Cutinases are well known in the art and are available from a widevariety of sources. See Cutinases from Fungi and Pollen, P. E.Kolattukudy, pg. 472-504, incorporated herein by reference, fordiscussion of cutinases useful in the practice of the invention. Apreferred cutinase is that cutinase isolated in a substantially pureform from Pseudomonas putida, particularly the P. putida, ATCC 53552,described in copending U.S. patent application, Ser. No. 932,959 filedNo. 19, 1986 and incorporated herein by reference, which enzymetherefrom has the following amino acid sequence: ##STR1## Other sourcesof bacterial and fungal cutinases include:

Fusarium solani pisi

Fusarium roseum sambucinum

Fusarium roseum culmorum

Helminthosporum sativum

Ulocladium consortiale

Streptomyces scabies

Colletotrichum capsici

Phytopthora cactorum

Botrytis cineria

Colletotrichum gloeosporioides

The cutinase of the invention should preferably be selected to cause atleast about 10%, and preferably 20%, hydrolysis of the given fat undergiven conditions. Normally the amount would be in a concentration offrom about 0.01% to about 5.0% by weight of the surfactant, andpreferably from about 0.05% to about 3%, such that upon dilution in washwater it is in a concentration of at least about 0.05 mg/L. Further, oneskilled in the art could take the preferred cutinase or, for thatmatter, any cutinase of the invention or any immuniologically identicalcutinase and use random or selective replacement of amino acids toproduce other cutinases which are more or less selective toward givensubstrates or include modification in activity such as oxidativestability. Substantially pure cutinase includes the isolated enzyme aswell as the broth containing the enzyme in unpurified form butessentially free of other enzymes and enzyme sources.

The natural substrate of cutinase is cutin which is a biopolyesterpolymer which covers the plant leaves, fruits, etc., see Structure,Biosynthesis and Biodegradation of Cutin and Suberin. (1981), P. E.Kolattukudy, Ann. Rev. Plant Physiol., 32, pgs. 539-567. Stainscomprising lipids which could be hydrolyzed or bound by cutinase on asubstrate such as cloth would be similar to the natural substrate cutin.Cutinase, for these types of stains, will be more effective than theprior art lipases. The cutinases will work especially well on gravy,oils and greases, plant or grass, oil based makeup and collar stains.

Cutiniases are distinguishable from other lipases by methods well knownin the art, See R. E. Purdy and P. E. Kolattukudy, Biochemistry,"Cutinase Assay", 14:2831-2840, (1975). Microbial cutinases from bothfungal and bacterial sources have very good activity at pH 8-pH 11 whichis an ideal pH condition for detergent use.

Because of the specific activity of cutinases, it is a preferred aspectof the invention to combine one or more other cutinases, or one or moreother enzymes, such as proteases, amylases or other lipases, along withthe cutinase of the invention in the cleaning composition. Further,Applicant shows a synergistic increase in hydrolytic activity ofcutinase when two or more surfactants are combined along with thecutinase enzyme.

Cutinases then are ideal for cleaning composition inclusion. They havestability oxidatively such as in H₂ O₂. They have good stability in atemperature range of from about 20°-50° C. which is ideal from acleaning point of view. They are also stable in the presence of otherenzymes; e.g., proteases, and as such, are ideal for mixtures ofenzymes.

The Surfactant

A number of known compounds are suitable surfactants useful in thepresent compositions. These include nonionic, anionic, cationic, orzwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 toBarry J. Anderson and U.S. Pat. No. 4,261,868 to Jiri Hora et al. Theart is familiar with the different formulations which can be used ascleaning compositions.

The Cleaning Compositions and Method of Use

Cutinases can be formulated as a purposefully added ingredient intoknown powdered and liquid detergents having pH between 6.5 and 12.0 atlevels of about 0.01 to about 5% (preferably 0.05 to 0.5%) by weight ofthe detergent. These detergent cleaning compositions can also includeother enzymes such as known proteases and amylases, as well as bleaches,colorants, builders, and stabilizers.

The cutinase of the invention may be added to powdered detergents in theform of granulates or prills, prepared by methods known in the art suchas described in British Patent Nos. 1,324,166 and 1,362,365 and U.S.Pat. Nos. 3,519,570; 4,106,991 and 4,242,219.

The cutinase preparations of the invention can be prepared bycultivating the microorganisms defined herein or otherwise cutinasecontaining microorganism under appropriate conditions. In order toobtain reasonable yields of enzyme, media containing readily assimilablecarbon and energy sources as necessary such as a nitrogen source, aswell as calcium and magnesium salts and trace elements and cutin, ormonomers of cutin, or compounds resembling cutin or cutin monomers. Onecould also obtain the gene for cutinan and express in any organism ofchoice where one may not have to add cutin or cutin monomers into thefermentation.

The addition of cutinase to conventional cleaning compositions does notcreate any special use limitation. In other words, any temperature andpH suitable for detergent compositions containing enzymes is alsosuitable for the present compositions.

Although the preferred form of the invention has been described above,it will be obvious to those skilled in the art to which the inventionpertains, that, after understanding the invention and in view of thefollowing testing as a whole, various changes and equivalentmodifications may be made without parting from the scope of theinvention as defined by the claims.

STABILITY CUTINASE AGAINST PROTEASES

Reaction conditions:

Buffer: 0.1 M NaP, pH 10

Temp: 37° C.

Lipase: 42 ug/ml

Approximately 1:1 protease:cutinase aqueous solution were made up withthe following results.

ENZYME ACTIVITY

    ______________________________________                                        Protease                                                                      Incubation Time                                                                            0 min   5 min   10 min                                                                              15 min                                                                              14 hrs                               ______________________________________                                        None         1.57    1.60    1.47  1.63  1.61                                 Maxacal (35 μg/ml)                                                                      1.68    1.58    1.72  1.66  0.134                                Esperase (64 μg/ml)                                                                     1.73    1.64    1.59  1.51  0.456                                ______________________________________                                    

Maxical is Gist-Brocade's brand of subtilisin enzyme (protease) Esperaseis Novo's brand of protease enzyme (protease)

TEMPERATURE STABILITY OF BACTERIAL CUTINASE

    ______________________________________                                        HALF LIFE AT 50° C.                                                            pH   Hrs.                                                             ______________________________________                                                7    30                                                                       8    25                                                                       9    12                                                                       10     0.3                                                            ______________________________________                                    

Enzyme was incubated at 50° C. in 0.1M sodium phosphate buffer atvarious pH's and activity was measured by hydrolysis of trioctanoin inpolyvinyl alcohol emulsions.

EFFECT OF DETERGENTS ON HYDROLASE ACTIVITY

Reaction Conditions

substrate: p-nitro-phenyl butyrate, 1 mm (pnp)

pH: 8.0

buffer: 0.1m tris pH 8.0

temperature: 25° C.

enzyme: bacterial cutinase from ATCC 53552

An aqueous solution with the following were made up and the enzymeactivity was measured in these solutions using pnp as a substrate byfollowing absorbance of p-nitrophenol at 410 mm.

    ______________________________________                                                      SDS %                                                           Triton x-100 (octoxynol)                                                                    (sodium dodecyl sulfate)                                                                      % Activity                                      ______________________________________                                        0             0               100                                             0.2           --              78                                              0.4           --              60                                              --             0.05           30                                              --            0.1             23                                              --            0.2             14                                              --            0.4              6                                              0.4           0.4             78                                              0.2           0.2             98                                              0.2            0.05           125                                             0.2           0.1             138                                             6.1           0.1             130                                              0.05          0.05           132                                             ______________________________________                                    

(1) Non-ionic detergent inhibition is not significant at lowconcentrations.

(2) An ionic detergent inhibitor at high concentrations.

(3) Mixture of anionic and non-ionic detergents stimulate activity.

STABILITY TOWARD OXIDANTS

Cutinase 0.5 mg/ml in 0/1m sodium phosphate buffer, was incubated withvarious levels of hydrogen peroxide at pH 8.4 and 25° C. for 2 hours,and hydrolytic activity was measured by a pH-stat usingtrioctanoim-polyvinyl alcohol emulsion.

    ______________________________________                                                     Hydrolytic Activity                                              [H.sub.2 O.sub.2 ppm]                                                                      % Remaining                                                      ______________________________________                                         0           100                                                              100          86                                                               200          86                                                               500          91                                                               1000         95                                                               ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                         ##STR2##                                                                     ______________________________________                                         □ Hydrolase activity.                                         

                  TABLE 2                                                         ______________________________________                                         ##STR3##                                                                     ______________________________________                                         □ Hydrolase activity.                                         

What is claimed is:
 1. An improved method for enzymatically cleaning amaterial having a cutin stain comprising:(a) forming an aqueous solutioncomprising a cutinase enzyme derived from Pseudomonas putida ATCC 53552having a concentration of said enzyme in said solution of from about0.05 mg/l to about 100 mg/L, and a surfactant combination of sodiumdodecyl sulfate and octoxynol wherein said sodium dodecyl sulfate iscontained in said aqueous solution in a concentration of from about 0.05to about 0.1 percent and further wherein said octoxynol is contained insaid aqueous solution in a concentration of from about 0.05 to about 0.2percent; (b) contacting the material to be cleaned with the aqueoussolution of step (a); and (c) rinsing the material of step (b).